: The peripheral primary B cell compartment in mammals is characterized by expression of antigen receptors (BCRs) with "multi-reactivity" for both foreign and autoantigens. During the development of B cell memory the BCRS expressed by responding clonotypes are extensively altered by V gene somatic hypermutation. Hypermutation can create autoreactivity de novo, or enhance the autoreactivity intrinsic to precursor BCRS. We and others have proposed that a stringent self-tolerance process operates on B cells differentiating to the memory phenotype during foreign antigen driven immune responses. In the previous funding period we obtained evidence supporting this hypothesis. A B cell clonotype that dominates the memory compartment of the anti-arsonate immune response of A/J mice expresses BCRs with affinity for both arsonate and the self antigen DNA. In contrast, the hypermutated derivatives of these BCRs expressed by the memory compartment display increased affinity for arsonate and no affinity for DNA. However, if B cell apoptotic pathways are perturbed via enforced expression of Bcl-2 from a transgene, the BCRs expressed by members of this clonotype that enter the memory compartment display increased affinity for both arsonate and DNA. These data have led us to formulate the "specificity maturation" hypothesis-that during a foreign antigen driven immune response precursors to memory B cells undergoing hypermutation are subject to iterative cycles of both positive selection (for increased or unaltered affinity for the driving foreign antigen) and negative selection (for affinity for self antigens). The end result is a memory B cell compartment expressing BCRS with high specificity for the driving foreign antigen-a compartment that would efficiently recognize the foreign antigen and pose little risk for the development of autoimmunity. In the next proposed application period, we wish to test several tenets of this hypothesis as well as to begin to gain insight into the mechanism(s) that result in the generation of a memory B cell compartment with a high degree of specificity for the driving foreign antigen. Three interrelated questions will be addressed: 1) can B cells not subjected to central tolerance mechanisms and whose BCRs display "multi-reactivity," including autoreactivity, be recruited into the primary immune response and germinal center (GC) reaction, despite displaying a "self antigen regulated" phenotype?; 2) are members of such clones subsequently "purged" of their autoreactivity via somatic hypermutation or receptor editing prior to entry into the memory compartment?; and, 3) is the primary microenvironmental locale in which specificity maturation takes place the GC?